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transformMaizeGFFannotation.md

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Here I am showing how to transform the GFF file from B73 v3 reference to the Mo17 CAU assemblies. In this example the minimap2 splice aware sequence alignment model is used, which is much much faster than Mummer

Download the files

# B73 genomic sequence
wget ftp://ftp.ensemblgenomes.org/pub/plants/release-31/fasta/zea_mays/dna/Zea_mays.AGPv3.31.dna.genome.fa.gz

# GFF3 file for B73 V3 genome
wget ftp://ftp.ensemblgenomes.org/pub/plants/release-31/gff3/zea_mays/Zea_mays.AGPv3.31.gff3.gz

# Genomic sequences Mo17
https://ftp.maizegdb.org/MaizeGDB/FTP/Zm-Mo17-REFERENCE-CAU-1.0/Zm-Mo17-REFERENCE-CAU-1.0.fa.gz

# uncompress all the files
gunzip Zea_mays.AGPv3.31.dna.genome.fa.gz
gunzip Zea_mays.AGPv3.31.gff3.gz
gunzip Zm-Mo17-REFERENCE-CAU-1.0.fa.gz

get the transcripts sequence of B73

gean gff2seq -i Zea_mays.AGPv3.31.gff3 -r Zea_mays.AGPv3.31.dna.genome.fa -p b73_protein.fa -c b73_cds.fa -g b73_gene.fa

to save computational time, only the primary transcripts sequence are used

perl script/getPrimaryTranscriptSequenceFromGeanOutput.pl Zea_mays.AGPv3.31.gff3 b73_cds.fa >primary_cds.fasta

align the primary transcript CDS sequence to the Mo17 genome sequencing using minimap2

minimap2 -ax splice -t 90 -a -uf -C5 Zm-Mo17-REFERENCE-CAU-1.0.fa primary_cds.fasta >cds.sam

begin to run gean

split -l 20 cds.sam minimap2
ls minimap2* | awk '{print "gean spltogff -i Zea_mays.AGPv3.31.gff3 -l 100000 -w 5000 -r Zea_mays.AGPv3.31.dna.genome.fa -a "$1" -s Zm-Mo17-REFERENCE-CAU-1.0.fa -o "$1".gff"}' > spltogff_commnd
xjobs -j 50 -s spltogff_commnd

then merge those files together

cat minimap*gff > mo17.gff

do some duplication filtering

gean purifygff -i mo17.gff -s Zm-Mo17-REFERENCE-CAU-1.0.fa -o mo17_purify.gff

keep only very confident non-redundant records by syntenic analysis

gean sinsyn -i b73.gff -s Mo17.fa -a mo17.gff -o Mo17_sinsyn.gff

keep only those transcript records only with ORF conserved

gean orf -i Mo17_sinsyn.gff -s Mo17.fa -o Mo17_sinsyn_orf.gff
this is pipeline to transform the gene structure annotation from reference genome sequence to another de novo assembly genome squence. This is not a complete pipeline to annotate the new assembly.
If you are doing genome assembly for a new accession using different genome sequence assembly pipelines. The number of successfully transformed transcripts (marked as ConservedFunction in the output gff file) could be used to measure the genome assembly quality.